Sodium channel proteins embedded in cellular membranes of muscle cells, neurons and other excitable cells help produce and propagate electrical impulses and are implicated in many human diseases and conditions. Sodium channels are often composed of a pore-forming α subunit, having four homologous domains DI-DIV and six transmembrane regions S1-S6 per domain, and at least three auxiliary subunits β1, β2 and β3. The α subunit is sufficient to form a functional channel for generating sodium current flow across cellular membranes. An extensive review of cardiac ion channels published in Annual Review of Physiology 64:431-75 (2002) is incorporated by reference in its entirety as if set forth herein.
Human cardiac sodium channels play a critical role in cardiac excitation. hNav1.5, a human cardiac sodium channel α subunit encoded by the SCN5A gene forms a functioning monomeric sodium channel that carries the inward Na current (INa) in the heart. The INa current is vital for excitation and conduction in working myocardium and in specialized conduction tissue such as Purkinje fibers.
Three distinct full-length polymorphic SCN5A clones that encode the hNav1.5 human cardiac sodium channel (designated SCN5A hH1, SCN5A hH1a, and SCN5A hH1b (or simply hH1, hH1a, and hH1b, respectively) have been isolated from human cardiac cDNA libraries (Gellens, M. E. et al., “primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel,” Proc. Natl. Acad. Sci. U.S.A. 89, 554-558 (1992); Hartmann, H. A. et al., Effects of III-IV linker mutations on human heart Na+ channel inactivation gating. Circ. Res. 75, 114-122 (1994), Ye, B. and J. Makielski, Third Complete Sequence of Human Cardiac Sodium Channel α Subunit Reveals Polymorphism in Domain I and II, Biophys. J. 80(1):225c (2001), each incorporated by reference herein as if set forth in its entirety.).
Subsequent to the publication of the hH1b sequence by Ye and Makielski, the authors determined errors in the published SCN5A hH1 protein sequence. The true polymorphisms among hH1, hH1a and hH1b are reflected in Table 1, infra. The amino acid numbering follows that of the original hH1 clone which contains 2016 amino acids. All of the differences are confined to the cytoplasmic linkers between DI-II and between DII-III. Briefly, hH1 and hH1a differ by just 3 amino acids—T559 vs. A559, Q1027 vs. R1027, and Q1077 vs. Q1077de1 (hH1 vs. hH1a, respectively)—over a total length of 2016/2015 amino acids, respectively. The hH1b protein also differs from either hH1 or hH1a at positions 559, 1027 and 1077, as well as at positions 558 and 618. The arginine at position 558 in hH1b is consistent with a previously characterized histidine-to-arginine polymorphism (Iwasa, et al., “Twenty single nucleotide polymorphisms (SNPs) and their allelic frequencies in four genes that are responsible for familial long QT syndrome in the Japanese population,” J. Hum. Genet. 45, 182-183). The isoleucine at position 618 is consistent with a known high-frequency spontaneous conservative leucine-to-isoleucine substitution.
The significance of polymorphisms in the sodium channel is still unknown. For example, it is not known how such polymorphisms affect the mutation phenotype of SCN5A. Nonetheless, identified polymorphisms can help identify disease-associated mutations in SCN5A. For example, various SCN5A mutations are associated with congenital Long QT syndrome, idiopathic ventricular fibrillation and the Brugada syndrome (Keating and Sanguinetti 2001).
In separate studies, the two known polymorphic forms showed only minor kinetic differences that can be attributed to different expression systems and study techniques including solutions, temperature, and protocols. (Gellens, M. E. et al., supra; Hartmann et. al., supra; and Wattanasirichaigoon et. al. 1999). Subtle differences in kinetics such as decay rates, inactivation midpoints, and late INa, however, may be important in controlling repolarization.
Sodium channel α subunits encoded by an SCN5A hH1a clone carrying an arrhythmogenic missense methionine-to-leucine mutation at amino acid 1766 (M1766L) further exhibit a significant inward sodium current level drop, relative to the current level in channels encoded by a wild type hH1a clone. Recently, M1766L in the hH1a background was shown to have a trafficking defect and to cause QT prolongation and ventricular arrhythmia. These conditions can be rescued by low temperature, antiarrhythmic drugs and the β1 subunit. Valdivia et. al., C. R. et al., A Novel SCN5A Arrhythmia Mutation M1766L with Expression Defect Rescued by Mexiletine,” Cardiovasc. Res. 54(3):624-9 (2002).
In another aspect, drugs that can alter sodium channel activities can relieve or prevent symptoms of certain conditions such as cardiac arrhythmias. Cardiac arrhythmias are abnormalities in the rate, regularity, or site of origin of the cardiac impulse, or a disturbance in conduction of the impulse that alters the normal sequence of atrial or ventricular activation. One known way to treat cardiac arrhythmias is to block the activity of a cardiac sodium channel. Sodium channel blockers used to treat cardiac arrhythmias include: Quinidine, Lidocaine, Procainamide, Mexiletine, Flecainide, Moricizine, and Disopyramide. Identifying other polymorphic forms of human cardiac sodium channel will advance our understanding of sodium channel-related heart problems and provide new tools for developing diagnostic, prophylactic and therapeutic strategies.
The art is uncertain as to whether any of the three polymorphic SCN5A isolates encodes a standard or reference hNav1.5 protein. This uncertainty precludes a well-reasoned analysis of mutations at particular amino acid residues in a consistent background. It is important to employ channel proteins having a suitable genetic background when evaluating one or more mutations of interest so that the actual effect of the mutations is noted. In addition, when studying possible direct and indirect drug interactions with cardiac channels, it is important to assess those interactions in the proper context. The full import of the genetic background of mutations in hNav1.5 has not heretofore been understood.